Systems biology of tomato fruit development: combined transcript, protein, and metabolite analysis of tomato transcription factor (nor, rin) and ethylene receptor (Nr) mutants reveals novel regulatory interactions.

Tomato (Solanum lycopersicum) is an established model to study fleshy fruit development and ripening. Tomato ripening is regulated independently and cooperatively by ethylene and transcription factors, including nonripening (NOR) and ripening-inhibitor (RIN). Mutations of NOR, RIN, and the ethylene receptor Never-ripe (Nr), which block ethylene perception and inhibit ripening, have proven to be great tools for advancing our understanding of the developmental programs regulating ripening. In this study, we present systems analysis of nor, rin, and Nr at the transcriptomic, proteomic, and metabolomic levels during development and ripening. Metabolic profiling marked shifts in the abundance of metabolites of primary metabolism, which lead to decreases in metabolic activity during ripening. When combined with transcriptomic and proteomic data, several aspects of the regulation of metabolism during ripening were revealed. First, correlations between the expression levels of a transcript and the abundance of its corresponding protein were infrequently observed during early ripening, suggesting that posttranscriptional regulatory mechanisms play an important role in these stages; however, this correlation was much greater in later stages. Second, we observed very strong correlation between ripening-associated transcripts and specific metabolite groups, such as organic acids, sugars, and cell wall-related metabolites, underlining the importance of these metabolic pathways during fruit ripening. These results further revealed multiple ethylene-associated events during tomato ripening, providing new insights into the molecular biology of ethylene-mediated ripening regulatory networks.

A secreted effector protein (SNE1) from Phytophthora infestans is a broadly acting suppressor of programmed cell death.

Evasion or active suppression of host defenses are critical strategies employed by biotrophic phytopathogens and hemibiotrophs whose infection mechanism includes sequential biotrophic and necrotrophic stages. Although defense suppression by secreted effector proteins has been well studied in bacteria, equivalent systems in fungi and oomycetes are poorly understood. We report the characterization of SNE1 (suppressor of necrosis 1), a gene encoding a secreted protein from the hemibiotrophic oomycete Phytophthora infestans that is specifically expressed at the transcriptional level during biotrophic growth within the host plant tomato (Solanum lycopersicum). Using transient expression assays, we show that SNE1 suppresses the action of secreted cell death-inducing effectors from Phytophthora that are expressed during the necrotrophic growth phase, as well as programmed cell death mediated by a range of Avr-R protein interactions. We also report that SNE1 contains predicted NLS motifs and translocates to the plant nucleus in transient expression studies. A conceptual model is presented in which the sequential coordinated secretion of antagonistic effectors by P. infestans first suppresses, but then induces, host cell death, thereby providing a highly regulated means to control the transition from biotrophy to necrotrophy.

Structure of the glucanase inhibitor protein (GIP) family from phytophthora species suggests coevolution with plant endo-beta-1,3-glucanases.

During invasion of their plant hosts, species of the oomycete genus Phytophthora secrete glucanase inhibitor proteins (GIPs) into the plant apoplast, which bind and inhibit the activity of plant extracellular endo-beta-1,3-glucanases (EGases). GIPs show structural homology to the chymotrypsin class of serine proteases (SP) but lack proteolytic activity due to the absence of an intact catalytic triad and, thus, belong to a broader class of proteins called serine protease homologs (SPH). To study the evolutionary relationship between GIPs and functional SP, database searches were used to identify 48 GIP homologs in the P. sojae, P. ramorum, and P. infestans genomes, composing GIPs, SPH, and potentially functional SP. Analyses of P. infestans-inoculated tomato leaves showed that P. infestans GIPs and tomato EGases are present in the apoplast and form stable complexes in planta. Studies of the temporal expression of a four-membered GIP family from P. infestans (PiGIP1 to PiGIP4) further revealed that the genes show distinctly different patterns during an infection timecourse. Codon evolution analyses of GIP homologs identified several positively selected peptide sites and structural modeling revealed them to be in close proximity to rapidly evolving EGase residues, suggesting that the interaction between GIPs and EGases has the hallmarks of a coevolving molecular arms race.

Plant glycosyl hydrolases and biofuels: a natural marriage.

Much of what is currently known about the structure, properties and biochemical activities of glycosyl hydrolases (GHs) has resulted from detailed studies of microbial enzymes. Conversely, such information is sparse in the plant GH literature, where the focus has traditionally been on studying expression and biological function. However, the current resurgence of interest in lignocellulosic biofuels is catalyzing new interest in this field, and recent reports suggest that some plant GH families have more in common with their microbial counterparts than was previously suspected. The repertoires of plant GHs, with their associated catalytic activities and polysaccharide binding affinities, may have valuable applications in modifying plant cell wall architecture and in the development and characterization of new bioenergy feedstocks.

A functional screen to characterize the secretomes of eukaryotic pathogens and their hosts in planta.

Complex suites of proteins that are secreted by plants and phytopathogens into the plant apoplast play crucial roles in surveillance, assault, defense, and counter-defense. High-throughput genome-scale strategies are being developed to better understand the nature of these "secretomes" and the identity of pathogen-derived effector proteins that subvert plant defenses and promote pathogenicity. Although combined bioinformatic and experimental approaches recently have provided comprehensive coverage of secreted proteins from bacterial phytopathogens, far less is known about the secretomes and batteries of effectors of eukaryotic phytopathogens; notably fungi and oomycetes. The yeast secretion trap (YST) represents a potentially valuable technique to simultaneously target pathogen and host secretomes in infected plant material. A YST screen, using a new vector system, was applied to study the interaction between tomato (Solanum lycopersicum) and the oomycete Phytophthora infestans, revealing sets of genes encoding secreted proteins from both pathogen and host. Most of those from the oomycete had no identifiable function and were detectable in planta only during pathogenesis, underlining the value of YST as a tool to identify new candidate effectors and pathogenicity factors. In addition, the majority of the P. infestans proteins had homologs in the genomes of the related oomycetes R. sojae and P. ramorum.

Phytophthora genome sequences uncover evolutionary origins and mechanisms of pathogenesis.

Draft genome sequences have been determined for the soybean pathogen Phytophthora sojae and the sudden oak death pathogen Phytophthora ramorum. Oömycetes such as these Phytophthora species share the kingdom Stramenopila with photosynthetic algae such as diatoms, and the presence of many Phytophthora genes of probable phototroph origin supports a photosynthetic ancestry for the stramenopiles. Comparison of the two species' genomes reveals a rapid expansion and diversification of many protein families associated with plant infection such as hydrolases, ABC transporters, protein toxins, proteinase inhibitors, and, in particular, a superfamily of 700 proteins with similarity to known oömycete avirulence genes.

Sample extraction techniques for enhanced proteomic analysis of plant tissues.

Major improvements in proteomic techniques in recent years have led to an increase in their application in all biological fields, including plant sciences. For all proteomic approaches, protein extraction and sample preparation are of utmost importance for optimal results; however, extraction of proteins from plant tissues represents a great challenge. Plant tissues usually contain relatively low amounts of proteins and high concentrations of proteases and compounds that potentially can limit tissue disintegration and interfere with subsequent protein separation and identification. An effective protein extraction protocol must also be adaptable to the great variation in the sets of secondary metabolites and potentially contaminating compounds that occurs between tissues (e.g., leaves, roots, fruit, seeds and stems) and between species. Here we present two basic protein extraction protocols that have successfully been used with diverse plant tissues, including recalcitrant tissues. The first method is based on phenol extraction coupled with ammonium acetate precipitation, and the second is based on trichloroacetic acid (TCA) precipitation. Both extraction protocols can be completed within 2 d.

Digging deeper into the plant cell wall proteome.

The proteome of the plant cell wall/apoplast is less well characterized than those of other subcellular compartments. This largely reflects the many technical challenges involved in extracting and identifying extracellular proteins, many of which resist isolation and identification, and in capturing a population that is both comprehensive and relatively uncontaminated with intracellular proteins. However, a range of disruptive techniques, involving tissue homogenization and subsequent sequential extraction and non-disruptive approaches has been developed. These approaches have been complemented more recently by other genome-scale screens, such as secretion traps that reveal the genes encoding proteins with N-terminal signal peptides that are targeted to the secretory pathway, many of which are subsequently localized in the wall. While the size and complexity of the wall proteome is still unresolved, the combination of experimental tools and computational prediction is rapidly expanding the catalog of known wall-localized proteins, suggesting the unexpected extracellular localization of other polypeptides and providing the basis for further exploration of plant wall structure and function.